A stem cell must meet the following criteria: (1) ability of a conal stem cell population to self-renew; (2) ability of a clonal stem cell population to generate a new, terminally differentiated cell type in vitro; (3) Ability of a clonal stem cell population to replace an absent terminally differentiated cell population when transplanted into an animal depleted of its own natural cells.
The neonatal period in human development is characterized by the presence of “stem” cells with the potential to develop along multiple differentiation pathways. The terminal differentiation of these cells is determined by cytokine and hormonal cues which co-ordinate organogenesis and tissue architecture. Murine embryonic stem (ES) cells have been isolated and studied extensively in vitro and in vivo. Using exogenous stimuli in vitro, investigators have induced ES cell differentiation along multiple lineage pathways. These pathways include neuronal, B lineage lymphoid, and adipocytes (Dani et al. (1997) J. Cell Sci. 110:1279; Remoncourt et al. (1998) Mech Dev 79:185; O'Shea, S. (1999) Anat. Rec. 257:32, 1999). The ES cells have been manipulated in vivo by homologous recombination techniques to generate gene specific null or “knock-out” mice (Johnson, R. S. (1989) Science 245:1234). Once ES cell clones lacking a specific gene are isolated, they are transplanted into a fertilized murine zygote. The progeny of this isolated ES cell can develop into any and all murine tissues in a coordinated manner.
Multipotential stem cells exist in tissues of the adult organism. The best characterized example of a “stem cell” is the hematopoietic progenitor isolated from the bone marrow and peripheral blood. Seminal studies by Trentin and colleagues (Trentin (1965) Cardiovasc. Res. Cent. Bull 4:38; Till & McCulloch (1961) Rad. Res. 14:213) examined lethally irradiated mice. In the absence of treatment, these animals died because they failed to replenish their circulating blood cells; however, transplantation of bone marrow cells from syngeneic donor animals rescued the host animal. The donor cells were responsible for repopulating all of the circulating blood cells. A wealth of elegant studies have gone on to demonstrate that donation of a finite number of undifferentiated hematopoietic stem cells is capable of regenerating each of the eight or more different blood cell lineages in a host animal. This work has provided the basis for bone marrow transplantation, a widely accepted therapeutic modality for cancer and inborn errors of metabolism. Thus, hematopoietic stem cells remain present in normal human bone marrow throughout life; they are not limited to the neonatal period.
There is exciting new evidence that hematopoietic progenitors may not be limited to the bone marrow microenvironment. Investigators at the University of Calgary have examined neuronal stem cells, which routinely differentiate along neuronal cell lineage pathways. When these cells were transplanted into lethally irradiated hosts, the investigators detected the presence of donor cell markers in newly produced myeloid and lymphoid cells (Bjornson (1999) Science 283:534). Investigators at the Baylor College of Medicine have performed similar studies using satellite cells isolated from murine skeletal muscle (Jackson et al. (1999) PNAS 96:14482). When these muscle-derived cells were transplanted into lethally irradiated hosts, the investigators detected the presence of the muscle gene markers in all blood cell lineages. Together, these studies indicate that neuronal and muscle tissues contain stem cells capable of hematopoietic differentiation. This suggest that sites other than the bone marrow may provide a renewable source of hematopoietic progenitors with potential application to human disease therapy (Quesenberry et al. (1999) J. Neurotrauma 16:661: Scheffler et al. (1999) Trends Neurosci 22:348; Svendson & Smith (1999) Trends Neurosci 22:357).
Just as neuronal and muscle cells are capable of regenerating the irradiated bone marrow, bone marrow derived cells are capable of repopulating other organ sites. When bone marrow derived hematopoietic and stromal cells are transplanted into an animal with an injured liver, they are capable of regenerating hepatic oval cells in the host animal (Peterse et al. (1999) Science 284:1168). Similarly, when labeled bone marrow stromal cells are implanted into the lateral ventricle of a neonatal mouse, they were capable of differentiating into mature astrocytes (Kopen et al. (1999) PNAS 96:10711). Indeed, when bone marrow stromal cells are transplanted intraperitoneally into mice, they are detected throughout the organs of the host animal, including the spleen, lung, bone marrow, bone, cartilage, and skin (Pereira et al (1998) PNAS 95:p 1142, 1998). These studies suggest that the bone marrow stromal cell is capable of differentiating into lineages different from their original dermal origin (Kopen et a. (1999) PNAS 96:10711).
The recent development of entire organisms from a single donor cell is consistent with this hypothesis. For example, the “Dolly” experiment showed that cells isolated from an ovine mammary gland could develop into a mature sheep. In similar murine studies, cells derived from the corpus luteum of the ovary could develop into a mature mouse. These studies suggest that stem cells with ability to differentiate into any and all cell types continue to exist in the adult organism. Thus, “embryonic” stem cells may be retained throughout the life of an individual.
The adult bone marrow microenvironment is the potential source for these hypothetical stem cells. Cells isolated from the adult marrow are referred to by a variety of names, including stromal cells, stromal stem cells, mesenchymal stem cells (MSCs), mesenchymal fibroblasts, reticular-endothelial cells, and Westen-Bainton cells (Gimble et al.(1996) Bone 19:421, 1996). In vitro studies have determined that these cells can differentiate along multiple mesenchymal or mesodermal lineages which include, but are not limited to, adipocytes (fat cells) (Gimble et al. (1990) Eur J. Immunol 20:379), Chondrocytes (Bruder et al. (1994) J. Cell Biochem. 56:283), hematopoietic supporting cells (Pietrangeli et al. (1988) Eur. J. Immunol. 18:863), skeletal muscle myocytes (Prockop (1998) J. Cell Biochem Suppl. 30–31:284–5), smooth muscle myocytes (Charbord), and osteoblasts (Beresford et al. (1992) J. Cell Sci. 99:131; Dorheim et al. (1993) J. Cell Physiol. 154:317). In addition, bone marrow stromal cells display the ability to differentiate into astrocytes (Kopen et al. (1999) PNAS 96:10711) and hepatic oval cells (Petersen et al. (1999) Science 284:1168). Based on these findings, the bone marrow has been proposed as a source of stromal stem cells for regeneration of bone, cartilage, muscle, adipose tissue, liver, neuronal, and other tissues. However, extraction of bone marrow stromal cells presents a high level of risk and discomfort to the patient.
In contrast, adult human extramedullary adipose tissue-derived stromal cells represent a stromal stem cell source that can be harvested routinely with minimal risk or discomfort to the patient. Pathologic evidence suggests that adipose-derived stromal cells are capable of differentiation along multiple lineage pathways. The most common soft tissue tumors, liposarcomas, develop from adipocyte-like cells. Soft tissue tumors of mixed origin are relatively common. These tumors may include elements of adipose tissue, muscle (smooth or skeletal), cartilage, and/or bone. In patients with a rare condition known as progressive osseous heteroplasia, subcutaneous adipocytes form bone for unknown reasons.
Recent studies have demonstrated the specific ability of bone marrow-derived stromal cells to undergo neuronal differentiation in vitro (Woodbury et al. (2000) J Neuroscience Research 61:364; Sanchez-Ramos et al. (2000) Exp Neurology 164:247). In these investigations, treatment of bone marrow stromal cells with antioxidants, epidermal growth factor (EGF), or brain derived neurotrophic factor (BDNF) induced the cells to undergo morphologic changes consistent with neuronal differentiation, i.e., the extension of long cell processes terminating in growth cones and filopodia (Woodbury et al. (2000) J Neuroscience Research 61:364; Sanchez-Ramos et al. (2000) Exp Neurology 164:247). In addition, these agents induced the expression of neuronal specific protein including nestin, neuron-specific enolase (NSE), neurofilament M (NF-M), NeuN, and the nerve growth factor receptor trkA (Woodbury et al. (2000) J Neuroscience Research 61:364; Sanchez-Ramos et al. (2000) Exp Neurology 164:247
U.S. Pat. No. 5,486,359 to Osiris is directed to an isolated, homogeneous population of human mesenchymal stem cells which can differentiate into cells of more than one connective tissue type. The patent discloses a process for isolating, purifying, and greatly replicating these cells in culture, i.e. in vitro.
U.S. Pat. No. 5,942,225 to Case Western and Osiris describes a composition for inducing lineage-directed differentiation of isolated human mesenchymal stem cells into a single particular mesenchymal lineage, which includes human mesenchymal stem cells and one or more bioactive factors for inducing differentiation of the mesenchymal stem cells into a single particular lineage.
U.S. Pat. No. 5,736,396 to Case Western describes a method of inducing ex vivo lineage-directed differentiation of isolated human mesenchymal stem cells which includes contacting the mesenchymal stem cells with a bioactive factor so as to thereby induce ex vivo differentiation thereof into a single particular mesenchymal lineage. The patent also describes method of treating an individual in need of mesenchymal cells of a particular mesenchymal lineage which includes administering to an individual in need thereof a composition comprising isolated, human mesenchymal stem cells which have been induced to differentiate ex vivo by contact with a bioactive factor so as to thereby induce ex vivo differentiation of such cells into a single particular mesenchymal lineage.
U.S. Pat. No. 5,908,784 to Case Western discloses a composition for the in vitro chondrogenesis of human mesenchymal precursor cells and the in vitro formation of human chondrocytes therefrom, which composition includes isolated human mesenchymal stem cells condensed into close proximity as a packed cell pellet and at least one chondroinductive agent in contact therewith. The patent also describes a process for inducing chondrogenesis in mesenchymal stem cells by contacting mesenchymal stem cells with a chondroinductive agent in vitro wherein the stem cells are condensed into close proximity as a packed cell pellet.
U.S. Pat. No. 5,902,741 to Advanced Tissue Sciences, Inc. discloses a living cartilage tissue prepared in vitro, that includes cartilage-producing stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a three-dimensional framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. The patent also discloses a composition for growing new cartilage comprising mesenchymal stem cells in a polymeric carrier suitable for proliferation and differentiation of the cells into cartilage.
U.S. Pat. No. 5,863,531 to Advanced Tissue Sciences, Inc. discloses a tubular living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a three-dimensional tubular framework composed of a biocompatible, non-living material having interstitial spaces bridged by the stromal cells.
U.S. Pat. No. 5,811,094 to Osiris describes a method of producing a connective tissue which includes producing connective tissue in an individual in need thereof by administering to said individual a cell preparation containing human mesenchymal stem cells which is recovered from human bone marrow and which is substantially free of blood cells.
U.S. Pat. No. 6,030,836 describes a method of maintaining human hematopoietic stem cells in vitro comprising co-culturing human mesenchymal stem cells with the hematopoietic stem cells such that at least some of the hematopoietic stem cells maintain their stem cell phenotype.
U.S. Pat. No. 6,103,522 describes an irradiated immortalized human stromal cell line in a combined in vitro culture with human hematopoietic precursor cells.
WO 9602662A1 and U.S. Pat. No. 5,879,940 describes human bone marrow stromal cell lines that sustain hematopoiesis
U.S. Pat. No. 5,827,735 to Morphogen describes purified pleuripotent mesenchymal stem cells, which are substantially free of multinucleated myogenic lineage-committed cells, and which are predominantly stellate-shaped, wherein the mesenchymal stem cells form predominantly fibroblastic cells when contacted with muscle morphogenic protein in tissue culture medium containing 10% fetal calf serum and form predominantly branched multinucleated structures that spontaneously contract when contacted with muscle morphogenic protein and scar inhibitory factor in tissue culture with medium containing 10% fetal calf serum.
WO 99/94328 describes the use of mesenchymal stem cells to treat the central nervous system and a method of directing differentiation of bone marrow stromal cells.
WO 98/20731 to Osiris describes a mesenchymal megakaryocyte precursor composition and method of isolating MSCs associated with isolated megakaryocytes by isolating megakaryocytes.
WO 99/61587 to Osiris describes human CD45 and/or fibroblast and mesenchymal stem cells.
WO 00/53795 to the University of Pittsburgh and The Regents of the University of California discloses adipose-derived stem cells and lattices substantially free of adipocytes and red blood cells and clonal populations of connective tissue stem cells. The cells can be employed, alone or within biologically-compatible compositions, to generate differentiated tissues and structures, both in vivo and in vitro. Additionally, the cells can be expanded and cultured to produce hormones and to provide conditioned culture media for supporting the growth and expansion of other cell populations. In another aspect, WO 00/53795 a lipo-derived lattice substantially devoid of cells, which includes extracellular matrix material form adipose tissue. The lattice can be used as a substrate to facilitate the growth and differentiation of cells, whether in vivo or in vitro, into anlagen or mature tissue or structures. WO 00/53795 did not disclose adipose tissue derived stromal cells that have been induced to express at least one phenotypic characteristic of a neuronal, astroglial, hematopoietic progenitor, or hepatic cell in its priority document, nor did it disclose an isolated adipocyte tissue-derived stromal cell that has been dedifferentiated such that there is an absence of adipocyte phenotypic markers.
WO 99/28444 discloses methods and compositions for differentiating stromal cells form adipose tissue into cells having osteoblastic properties, and methods for improving a subject's bone structure.
WO 00/44882 discloses a method and composition for inducing stromal cells derived from adipose tissue into fully functional pleuripotent stem cells as evidenced by differentiated hematopoietic or blood cell lineages, neuronal lineages, epithelial lineages, and uses thereof.
It is a goal of the present invention to provide new cells and methods for therapeutic treatment, diagnosis, other medical uses, and other purposes, including the cellular production of desired materials.